Borrelia burgdorferi, the cauative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual genomic structure composed of a linear chromosome and a large number of linear and circular plasmids. Abundant evidence indicates that plasmid-encoded genes are critical for adaptation in the infectious cycle. In contrast to their essential role in vivo, plasmids are often lost during in vitro cultivation of B. burgdorferi. A major focus of our research is to determine the contributions of individual B. burgdorferi genes and plasmids at each stage of the infectious cycle, taking a molecular genetic approach. The genome of the type strain (B31) of B. burgdorferi has 12 linear and 9 circular plasmids, in addition to the linear chromosome. Previous studies from our lab and others have determined the roles of two linear plasmids, lp25 and lp28-1, in the mammalian host. The linear plasmid lp28-1 is required by B. burgdorferi to establish persistent infections in immunocompetent mice following needle inoculation, whereas lp25 is essential for B. burgdorferi growth in mammals under all conditions. Of the specific genes carried by these plasmids, others have shown that the bbe22 gene on lp25, encoding a nicotinamidase (PncA), is sufficient to restore infectivity in mammals to B. burgdorferi clones lacking lp25. In contrast, the essential gene or genes on lp28-1 for mouse infectivity have not been identified. However, the requirement in ticks for lp25 and lp28-1, or any of the genes they carry, has not been investigated. In a recent study, Grimm and colleagues analyzed the requirement for plasmids lp25 and lp28-1 by B. burgdorferi during colonization of ticks, a site where B. burgdorferi must survive and replicate to be maintained in its natural infectious cycle. This was accomplished by experimentally infecting ticks with wild type B. burgdorferi, with clones lacking lp28-1 or lp25, and with isogenic clones in which the respective plasmid had been restored. To do so, we utilized recently developed tools for the genetic manipulation of B. burgdorferi in combination with an efficient method for experimental tick infection. Many prior studies have examined the phenotypes of various B. burgdorferi clones lacking specific plasmids in mice, but it has been difficult to address their phenotypes in ticks, particularly when a clone is non-infectious in mice. We found that lp28-1 is not essential for survival and replication in the tick midgut, for migration to the salivary glands, or for transmission via the saliva to the mammalian host, although lp28-1 is required for establishing a persistent infection in mammals, regardless of the route of infection. In contrast, we found that lp25 is required for proficient infection of ticks and therefore is essential for persistence in both hosts of the infectious cycle. We also investigated the requirement for the nicotinamidase PncA by B. burgdorferi in the tick vector by infecting ticks with spirochetes that lack lp25, but contain the bbe22 gene (encoding PncA) on a shuttle vector. We found that complementation of B. burgdorferi lacking lp25 with just bbe22 restores the ability of spirochetes to colonize ticks, and infect mice after tick bite. Spirochetes lacking this enzyme presumably cannot utilize nicotinamide for the synthesis of NAD. These results indicate that B. burgdorferi requires bbe22 for infectivity in ticks but do not rule out the possibility that additional genes on lp25 may contribute to survival and replication of B. burgdorferi in the tick midgut. Further studies are needed to determine if any of the remaining genes on lp25, while not essential, provide an advantage for spirochete growth or survival in ticks and mice.This is the first study to investigate the requirement for specific plasmids by B. burgdorferi within the tick vector and it begins to establish the genomic components required for persistence of this pathogen throughout its natural infectious cycle.